Polymerase chain reaction (PCR), is a technology for rapidly amplifying DNA in vitro, and each cycle includes three steps: denaturation, annealing and extension. Firstly, a sample of double-stranded DNA is heated at a high temperature of about 95° C., and hydrogen bonds between the double strands are broken so that DNA is separated into two complementary single-stranded DNA molecules, and this process is referred to as a high-temperature melting reaction; then, the temperature is rapidly lowered to about 50-65° C. at which the single-stranded DNA binds to a primer according to the principle of complementary base pairing, which is called a low-temperature annealing reaction; after the annealing reaction, the temperature is rapidly raised to about 72° C. to permit extension reaction, where single nucleotides are sequentially added from the 3′end of the primer by DNA polymerase at an appropriate concentration of magnesium ion to form a new DNA. After such process, one original double-stranded DNA molecule becomes two new DNA molecules, and the number of DNA molecules is doubled. After each cycle, the number of target nucleic acid molecules is doubled, and these newly formed double-stranded molecules can be used as templates for the next cycle. After 30 to 40 cycles, the number of target nucleic acid molecules can increase to nearly 109 times. PCR is a method for obtaining a large number of target DNA segments in vitro, for further analysis and detection.
At present, the reaction devices for PCR mainly employ temperature-controlled metal blocks to heat PCR reaction tubes made of plastic, and by heating and cooling of the metal blocks to the equilibrium temperature, heat is transferred from the reaction tube to PCR reaction solution. The disadvantage of this device is that: the reaction volume is large, that is, the device usually has a large volume and heat capacity. Typically it takes 2-3 hours to complete a conventional PCR with 30 cycles, and most of the time is consumed by heating and cooling process, i.e., making the metal block to reach the equilibrium temperature and transferring heat from the reaction tube to the PCR reaction solution, therefore, fast and efficient PCR is difficult to be achieved.
In 2002, Madhavi Krishnan et al. reported a method named Rayleigh-Benard PCR (RB-PCR), based on the principle of heat conduction and thermal convection, using two constant temperature heat sources disposed at upper and lower regions respectively to establish a closed reaction cavity which has a temperature gradient from bottom to top, and thus convective motion of the PCR reagents occurs spontaneously, and makes the PCR reagents flow repeatedly through regions with different temperatures, to complete amplification. The amplification speed of this method is rapid, and the instrument is much simpler than traditional PCR instruments, but the amplification reagent should fill the entire closed cavity, resulting in difficulty in loading sample, and problems such as leakage and contamination.
Chou et al. in Taiwan University made improvements on the basis of the RB-PCR technology, by changing the closed reaction cavity to an open reaction tube with particular specification, and employing one single heat source of constant temperature to heat the bottom of the tube to drive the spontaneous circulation of reagents within the tube to complete amplification. This method solves the problem of leakage and contamination of RB-PCR.
However, common defects exist in the current amplification methods based on convective PCR, that is, flow path of the liquid in the tube is complicated. The flow path in the tube is a multilayer flow path nearly in the form of concentric ellipses (FIG. 1a). This complex multilayer flow path has the following problems in amplification:
1. Low Amplification Efficiency:
(a) Efficiency of denaturation: as shown in FIG. 1a, effective denaturation of templates or amplicons can occur when the templates or amplicons pass through region D1 where the temperature is not lower than the denaturation temperature required by the template or amplicon; while effective denaturation reaction cannot occur when the templates or amplicons pass through region D2 which is located higher than region D1, resulting in a low overall degeneration efficiency;
(b) Efficiency of annealing: As shown in FIG. 1a, effective annealing reaction may occur when the single-stranded templates and primers pass through region A1 where the temperature is not higher than the annealing temperature required by the templates and primers; while effective annealing reaction cannot occur when the single-stranded templates and primers pass through region A2 which is located lower than region A1, resulting in a low overall annealing efficiency
2. Poor Specificity of Amplification:
In the convection PCR, because of lack of an area and time period for annealing with a constant temperature, the temperature of the upper end of reaction tube is generally controlled to be lower than the annealing temperature of the primers by controlling the temperature field and flow field, to ensure a sufficient annealing of primers. However, when the single-stranded templates and (or) primers pass through a region where the temperature is too low in circulation, the specificity of annealing reaction is reduced, and non-specific pairing within one primer or between two primers, or between the primer and the template (or amplicon) may be formed easily, and as the extension reaction begins, non-specific amplification product is formed.
3. Differences Among the Parallel Amplification Reactions in Respective Tubes May Exist:
(a) As for qualitative detection at end-point of the reaction, the results mainly show the differences in amplification efficiency and product constitution: after denaturation, the non-specific amplification products as described in point 2 mentioned above will become the template in the next round of non-specific amplification, so that non-specific amplification is continuously enlarged, and will compete for primers, enzymes, dNTP and other reaction components availability with the correct amplification, resulting in inhibition of the correct amplification and reduction of the reaction efficiency. However, it is not known whether or when this non-specific reaction occurs, and the occurrence rate of this reaction is uncontrolled, that is, there is a certain randomness, which will cause inconsistency in term of the amplification efficiencies between reaction tubes wherein such non-specific amplification occurs. In the reaction tubes where the non-specific amplification occurs at an earlier time point or has a higher rate of occurrence, the reaction efficiency will be lower than that in the reaction tubes where the non-specific amplification occurs at a later time point or has a lower rate of occurrence. And in the two reaction tubes mentioned above, the reaction efficiencies are both lower than that in the reaction tubes where the non-specific amplification has not occurred. Similarly, there is a high proportion of non-specific products in the reaction tubes where the non-specific amplification occurs at an earlier time point or has a higher rate of occurrence, and there is a low proportion of non-specific products in the reaction tubes where the non-specific amplification occurs at an later time point or has a lower rate of occurrence, while in the reaction tubes where no non-specific amplification occurs, the products in tube are all correct amplification products.
(b) As for real-time quantitative detection, the results mainly show the differences in amplification efficiency per unit time: that is to say, real-time quantitative detection cannot be performed. As described above in points 1 and 2, when convective PCR reaction starts, it is not known if the double-stranded template can pass through the effective region for denaturation, or if single-stranded template and primer can pass through the effective region for annealing, and if non-specific reaction occurs during the annealing reaction. Therefore, at beginning of the reaction, differences in product constitution may be generated between different tubes. These differences not only may lead to problems described in the above 3(a), but also lead to differences in amount of products (templates) in different reaction tubes per unit time, and further result in differences in the time point when the exponential phase of amplification is entered, therefore, quantification of templates cannot be performed by traditional real-time monitoring method.